Project Summary/ Abstract: The goal of this project is to create a tissue engineered vascularized skeletal muscle construct. Volumetric muscle loss (VML) is a muscle defect that impacts greater than 20% of the volume of an individual muscle, overwhelming the natural repair mechanisms and leading to chronic functional deficits in the affected muscle. Current treatment options for VML are limited by donor site morbidity, lack of donor tissue, and the need for a highly skilled surgical team. Developing a tissue engineered vascularized skeletal muscle construct would mitigate these complications and provide a clinically-relevant treatment for VML. An ideal cell source for skeletal muscle tissue engineering, adipose-derived stem cells (ASCs) have high proliferation rates, can directly differentiate into myoblasts, and evade the host immune system. ASCs will be grown on novel electrospun fibrin fibers that mimic the environment of native muscle by providing a muscle-like stiffness and topographic alignment cues to encourage ASC myogenesis in 3D. The fibers provide a number of advantages: they mimic the structure and mechanics of native muscle, are composed of a biological material yet can be manufactured reproducibly, and they integrate well with host tissue causing minimal fibrosis. The objective of the proposed study is to effectively overcome two major limitations associated with ASC-mediated tissue engineered skeletal muscle and enable functional regeneration of a VML defect. First, the efficiency of ASC differentiation into skeletal myoblasts will be increased. ASCs are a highly heterogeneous population and narrowing ASCs to a potentially pro-myogenic CD34+CD31- subpopulation in a myomimetic 3D microenvironment may result in significantly higher levels of myoblast differentiation, which will be necessary to obtain a contractile muscle graft. Second, a strategy to rapidly vascularize the tissue engineered constructs to maximize post-transplantation cell survival and integration of the engineered graft with the host tissues will be developed. Native skeletal muscle is heavily vascularized and increasing vasculature within muscle defects has been shown to improve muscle regeneration. Vascularized muscle constructs will be developed by bundling ASC-derived skeletal muscle grafts with fibers containing engineered vascular networks comprised of ASCs and endothelial progenitor cells (EPCs). The development of a vascularized muscle construct is likely to further enhance the in vivo regenerative potential of ASCs and their clinical translatability. In Specific Aim 1 the myogenic capabilities of CD34+CD31- ASC subpopulation versus unsorted ASCs on electrospun fibrin fibers will be compared. In Specific Aim 2 a 3D vascularized fibrin construct will be developed in vitro and its incorporation into host vasculature will be investigated in vivo. In Specific Aim 3 a vascularized muscle construct will be developed in vitro and its ability to enhance in vivo muscle regeneration in a VML defect will be assessed. This work will enable the development of a vascularized skeletal muscle construct with easily procurable cell types and off-the-shelf biomaterials that would provide a clinically-relevant treatment for VML.